This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. During every cell division, the replicated genome must be distributed accurately to daughter cells. Cells use multiple mechanisms to ensure that chromosomes are segregated with high fidelity by the microtubule-based mitotic spindle. One key checkpoint involves the kinase Aurora B, which destabilizes defective kinetochore-microtubule attachments that are not under tension. Aurora B is a member of the chromosomal passenger complex (CPC), a set of interacting proteins named for their localization to the centromere region of chromosomes through metaphase and subsequent relocalization to the central spindle at anaphase. We are studying the regulation of the CPC by post-translation modification in the budding yeast Saccharomyces cerevisiae. We will identify of post-translational modifications by mass spectroscopy of purified CPC phosphorylated by candidate kinases in vitro. Thus far we have identified a number of potential self-regulatory phosphorylation sites on the activator of Aurora B, INCENP. Future work will explore the function of these sites and identify modifications by other regulatory kinases such as Mps1 and Cdk1. This approach will give insight into the local signaling mechanisms that operate during cell division to ensure the coordinated alignment and segregation of all of the chromosomes in a cell.